title: PTHrP in differentiating human mesenchymal stem cells: transcript isoform expression, promoter methylation, and protein accumulation.
abstract: Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5' and 3' ends and the existence of three different transcriptional promoters (P1, P2 and P3), two of which (P2 and P3) contain CpG islands. It is known that the expression of PTHrP isoforms may be differentially regulated in a developmental stage- and tissue-specific manner. To search for novel molecular markers of stemness/differentiation, here we have examined isoform expression in fat-derived mesenchymal stem cells both maintained in stem conditions and induced toward adipo- and osteogenesis. In addition, the expression of the splicing isoforms derived from P2 and P3 promoters was correlated to the state of methylation of the latter. Moreover, we also performed a quantitative evaluation of intracellular and secreted PTHrP protein product in undifferentiated stem cells and in parallel cultures at various differentiation stages. The data obtained indicate that from the stemness condition to that of osteo- and adipo-genic differentiated cells, the expression of isoforms becomes increasingly selective, thereby being a potential gene signature for the monitoring of cell stem or committed/differentiating state and that the switching-off of PTHrP isoform expression is mostly promoter methylation-dependent. Moreover, PTHrP intracellular retention is down-regulated in osteo-differentiating cells whereas the secretion of the protein in the extracellular medium is up-regulated with respect to stem cells, thereby suggesting that these variations of the intracellular and extracellular levels of PTHrP could potentially be enclosed in the list of the available protein signature of osteogenic differentiation.
authors.affiliation: Dipartimento STEBICEF, Università di Palermo, Palermo, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Longo
authors.forename: Mariangela
authors.initials: M
authors.lastname: Librizzi
authors.forename: Flores
authors.initials: F
authors.lastname: Naselli
authors.forename: Fabio
authors.initials: F
authors.lastname: Caradonna
authors.forename: Edda
authors.initials: E
authors.lastname: Tobiasch
authors.forename: Claudio
authors.initials: C
authors.lastname: Luparello
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D017398:Alternative Splicing; D002454:Cell Differentiation; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D005091:Exons; D005786:Gene Expression Regulation; D006801:Humans; D007438:Introns; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D010011:Osteocytes / Q000166:cytology / Q000378:metabolism*; D044162:Parathyroid Hormone-Related Protein / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D044162:Parathyroid Hormone-Related Protein; D020033:Protein Isoforms
keywords: 3,3′,5,5′-tetramethylbenzidine; 5-bromo-4-chloro-3′-indolyphosphate; Adipogenesis; BCIP; BMP; D-PBS; Dulbecco's phosphate buffered saline; EDTA; FCS; Gene expression; MICE; MSC; MSRE; Mesenchymal stem cells; NBT; Osteogenesis; PCR; PMSF; PTHrP; Promoter methylation; SDS; SM; TBS-T; TMB; Tris-buffered saline-Tween 20; bone morphogenetic protein; dNTPs; deoxynucleotide triphosphates; ethylenediaminetetraacetic acid; fetal calf serum; mesenchymal stem cells; methylation-sensitive restriction endonuclease; microtiter immunocytochemical ELISA assay; nitro-blue tetrazolium; parathyroid hormone-related protein; phenylmethylsulfonyl fluoride; polymerase chain reaction; s.e.m.; semi-quantitative multiplex; sodium dodecyl sulfate; standard error of the mean